Whole-cell-mediated biotransformations

Metabolic pathways containing dioxygenases in wild-type strains are usually related to detoxification processes upon conversion of aromatic xenobiotics to phenols and catechols, which are more readily excreted. Within such pathways, the intermediate chiral cis-diol is rearomatized by a dihydrodiol-dehydrogenase. While this mild route to catechols is also exploited synthetically [221], the chirality is lost. In the context of asymmetric synthesis, such further biotransformations have to be prevented, which was initially realized by using mutant strains deficient in enzymes responsible for the rearomatization. Today, several dioxygenases with complementary substrate profiles are available, as outlined in Table 9.6. Considering the delicate architecture of these enzyme complexes, recombinant whole-cell-mediated biotransformations are the only option for such conversions. E. coli is preferably used as host and fermentation protocols have been optimized [222,223]. 

In contrast to 2,3-dioxygenases, the related ipso/ortho oxygenation of aryl carbox-ylates has received considerable less attention and has hardly been utilized by the synthetic community, so far. Biooxidation of benzoic acid and P-naphthalene carboxylate provide access to corresponding 1,2-dihydroxylated dihydroaryl compounds in excellent stereoselectivity (Scheme 9.35), analogous to TDO- and NDO-mediated ortho/meta oxygenations. Whole-cell-mediated biotransformations were performed with mutant strains of Rahtonia and Pseudomonas and enable access to preparative quantities in >5 gl titers [261,262]. 

To overcome this obstacle, two different approaches have been exploited, both possessing benefits and disadvantages. For the isolated enzyme, a closed-loop system has been developed, where an auxiliary substrate has been added to regenerate NAD(P)H. The second approach is based on whole-cell-mediated biotransformations. 

Several suitable whole-cell systems have been identified for deracemization biotransformations on a large diversity of substrates, as compiled recently [48]. In particular, heterocyclic alcohols were successfully converted by Sphingomonas [55]. Access to enantiocomplementaiy products was achieved with various strains of Aspergillus [56] or Rhizopus [57]. Biotransformations can even be accomplished with yacon and ginger [58]. Substrate titers were reported up to 8gl for Candida parapsUosis mediated biotransformations [59]. 

Enzyme-mediated chiral sulfoxidation has been reviewed comprehensively in historical context [188-191]. The biotransformation can be mediated by cytochrome P-450 and flavin-dependent MOs, peroxidases, and haloperoxidases. Owing to limited stability and troublesome protein isolation, a majority of biotransformations were reported using whole-cells or crude preparations. In particular, fungi have been identified as valuable sources of such biocatalysts and the catalytic entities have not been fully identified in all cases. 

Fig. 12 Biotransformation of levulinic acid to y-VL using whole cells from Pseudomonas putida, a thioesterase from E. coli (tesB) and extracytosolically expressed PONl. The cyclizatirai at a low pH value is mediated by the human paraoxonase PONl. From Martin et al. (2010)...

Though use of isolated purified enzymes is advantageous in that undesirable byproduct formation mediated by contaminating enzymes is avoided [37], in many industrial biotransformation processes for greater cost effectiveness the biocatalyst used is in the form of whole cells. For this reason baker s yeast, which is readily available, has attracted substantial attention from organic chemists as a catalyst for biotransformation processes. One of the first commercialized microbial biotransformation processes was baker s yeast-mediated production of (R)-phenylacetyl carbinol, where yeast pyruvate decarboxylase catalyzes acyloin formation during metabolism of sugars or pyruvate in the presence of benzaldehyde [38]. 

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